Discovery of RNA interference (RNAi) has reinvigorated interest in chemical modifications to optimize properties of small interfering RNAs (siRNAs). The long-term goal of our research is to explore RNA's structure and function using chemical approaches and to develop modifications for practical application in RNAi. The present proposal focuses on internucleoside amides as non-ionic mimics of the phosphodiester linkages and will test the hypotheses that amides (1) can be readily introduced in RNA using solid-phase synthesis, (2) are excellent mimics of the phosphate backbone of RNA, and (3) will increase enzymatic stability and cellular uptake of siRNAs without compromising their RNAi activity. We also envision that amides may improve biodistribution and pharmacokinetics of siRNAs. We propose an interdisciplinary (organic chemistry, structural biochemistry and RNA biology) study with the specific aims to: 1. Develop synthetic methods to introduce consecutive amide linkages at any desired location in RNA by adopting and optimizing solid-phase peptide, PNA, and RNA synthesis methods. 2. Confirm that amide-linked RNA can mimic the structure of natural RNA using UV spectroscopic and X- ray crystallographic techniques in collaboration with Prof. Martin Egli (Vanderbilt University). 3. Synthesize siRNAs having several amide linkages at the 3'-end of each strand and test their biological properties and RNAi activity in collaboration with Dr. Devin Leake (Dharmacon). Amides may offer several advantages for in vivo RNAi applications: (1) high nuclease resistance due to the absence of the natural phosphate;(2) enhanced cellular uptake due to the reduction of the negative charge;(3) improved biodistribution and pharmacokinetics due to the increased hydrophobicity. Despite these potentially beneficial properties, neither amides nor any other non-ionic linkages have been tested in RNAi. If accepted by RNAi proteins, amides may significantly improve properties of siRNAs and may be used to design a novel class of chemically modified siRNAs. Combination of synthetic chemistry, structural studies and RNA biology will provide unique insights into how chemical modifications (amides) influence conformation, hydration, and thermal stability of RNA. Such knowledge is important for rational design of nucleic acid analogues and for developing gene selective therapeutic agents for such long standing problems as cancer, viral infections, genetic disorders, and neurodegenerative diseases.